i) Field of the Invention
This invention relates to a human nucleic acid for cytidine deaminase that has been engineered into eukaryotic and bacterial expression vectors, to the expression of human cytidine deaminase by mammalian cells and bacterial cells, and to cells expressing human cytidine deaminase. The invention further relates to methods and gene therapies that employ the dominant selectable marker, cytidine deaminase, which has the ability to inactivate a toxic antimetabolite such as cytosine arabinoside by deamination to uracil arabinoside.
ii) Description of the Prior Art
Selectable markers are important tools in the study, regulation and function of genes and are potentially important in gene transfer therapies. Conferring a unique resistance to a cytotoxic agent enables the skilled artisan the ability to select genetically altered cells from a mixed population.
The enzyme cytidine deaminase (CD) has the ability to catalyze the deamination of cytosine arabinoside, an antimetabolite that is toxic to mammalian cells, to uracil arabinoside which is non-toxic at pharmacological concentrations (Chabner, et al., 1990, In Cancer Chemotherapy: Principles and Practice pp. 154-179, Lippincott Company). CD can also inactivate, by deamination, other cytosine nucleoside analogs that are currently used as anticancer agents (Chabot et al., 1983, Biochem. Pharmacol. 32: 1327-1328; Bouffard et al., 1993, Biochem. Pharmacol., 45: 1857-1861). In mammalian cells cytosine arabinoside (ARA-C), is metabolized to ARA-CMP, ARA-CDP and ARA-CTP, the latter nucleotide analog is incorporated into DNA, producing a potent inhibition of DNA synthesis and resulting in growth inhibition and cell death (Chabner, et al., 1990, In Cancer Chemotherapy: Principles and Practice pp 154-179, Lippincott Company).
The availability of a dominant selectable marker to cytosine analogs, would be a significant advantage for the skilled artisan in the field of molecular biology, cell biology and gene transfer technology in eukaryotes.
The efficacy of treatments of patients with cytosine nucleoside analogs, suffers from the relatively high doses of cytosine analogs required and the accompanying side effects resulting from normal cell cytotoxicity. Indeed, bone marrow suppression is the major dose-limiting toxicity produced by intensive chemotherapy with ARA-C and related cytosine nucleoside analogs.
It would thus be advantageous for the patient and clinician if susceptible cells, such as bone marrow cells could be protected from the toxic effects of the cytosine analog. This protection could permit an increase in the therapeutic index of the drug. In addition, it could permit an increase in the dose administered without substantially increasing the side effects of the drug. A similar approach has been reported for the establishment of methotrexate-resistant bone marrow cells (Corey, et al., 1990, Blood 75: 337-343).
A partial cDNA for human CD has been isolated and the expression of cytidine deaminase demonstrated in bacteria but not in eukaryotic cells (Kuhn, et al., 1993, Biochem. Biophys. Res. Com. 190: 1-7). Recently, the full-length human cDNA has been isolated and its DNA and amino acid sequence determined (Laliberte et al., 1994, Cancer Res. 54: 5401-5407). Having the full-length human cDNA for CD, it would be advantageous to engineer vectors permitting expression of cytidine deaminase in cells. It would also be beneficial to obtain bone marrow cells expressing human CD and implant them in a patient suffering form an immune disease, prior to the treatment of this patient with cytosine analogs. This kind of gene therapy and others, could be beneficial for the treatment of diseases including but not limited to acute T-cell disorders, rheumatoid arthritis, and autoimmune diseases. Gene therapy using CD could also be beneficial for preventing graft rejection.
Cytidine deaminase has been reported to inhibit the proliferation of myeloid hematopoietic cells (Boyum et al., 1994, Exp. Hematology, 22: 208-214). This enzyme could thus have therapeutic use in the treatment of certain types of leukemia.
It would be advantageous to obtain large amounts of human cytidine deaminase to test the therapeutic potential of the enzyme. In addition, it would be beneficial to obtain cells expressing CD, as they could be used to test for inhibitors or up-regulators of cytidine deaminase activity.